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Environmental Health Perspectives Vol. 75, pp. 81-86, 1987
The Role of^ Oncogenes^ in^ Chemical
Carcinogenesis
by S. Jill Stowers,* Robert R. Maronpot,t Steven H.
Reynolds,* and Marshall W. Anderson*
Proto-oncogenes are cellular genes that are expressed during normal growth and^ developmental^ pro- cesses. Altered versions of normal proto-oncogenes have been implicated in^ the development of human neoplasia. In this report, we show the detection of activated proto-oncogenes in various spontaneous and chemically induced rodent tumors. The majority of activated proto-oncogenes found in^ these^ tumors^ are members of the ras gene family and have been activated^ by a^ point mutation. Characterization^ of the activating mutation may be useful in^ determining whether this^ proto-oncogene was^ activated by^ direct interaction of the chemical with the DNA. Comparison of activating^ lesions^ in^ spontaneous^ versus^ chem- ically induced tumors should be helpful in^ determining whether^ the^ chemical^ acts via^ a^ genotoxic or a nongenotoxic mechanism. All^ of this information^ may be^ helpful^ in^ the^ assessment^ of^ potential carcin- ogenic hazards of human exposure to^ chemicals.
Introduction
Recent evidence^ suggests that^ neoplastic develop- ment, at least in part, is the result of the abnormal activation of a small set of cellular genes. These genes, termed proto-oncogenes, were^ originally discovered^ as the transduced (^) genes of acute transforming retrovi- ruses (1-3). Subsequent studies have established that these proto-oncogenes can also be activated as onco- genes by mechanisms^ independent^ of^ retroviruses^ (4). Mechanisms for the conversion of proto-oncogenes to activated oncogenes include point mutations, gene am- plification, chromosomal rearrangements, and promoter insertion (Fig. 1). The activation of proto-oncogenes by genetic alterations^ results^ in^ altered^ levels^ of^ expression of the normal protein product, or in normal or altered levels of expression of an abnormal protein. Proto-oncogenes are expressed during regulated growth such as embryogenesis, regeneration of dam- aged liver, and stimulation of cell mitosis by growth factors. Proto-oncogenes are highly conserved. They are detected in species as divergent as yeast, Droso- phila, and humans. Proto-oncogenes include genes that encode for growth factors (sis), growth factor receptors (neu, erbB, fms), regulatory proteins in^ signal trans- duction (ras family), nuclear regulatory proteins (myc,
*Laboratory of Biochemical Risk Analysis, National^ Institute^ of Environmental Health Sciences, P.O. Box^ 12233, Research^ Triangle Park, NC 27709. tChemical (^) Pathology Branch, National^ Toxicology Program, Na- tional Institute of Environmental Health^ Sciences, P.O.^ Box^ 12233, Research Triangle Park, NC 27709.
myb, fos),^ and^ tyrosine^ kinases^ (scr, abl, ras). Thus,
the encoded proteins appear to play a crucial role in normal cellular growth and/or differentiation. The activation of proto-oncogenes in spontaneous and
chemically induced tumors has been extensively studied
during the past several years. Although oncogenes such
as ras and myc can complement each other in the ma-
lignant transformation of a cell in vitro (2), the number
of proto-oncogenes that must be activated in the mul-
tistep process of carcinogenesis is unclear at present.
Also, new evidence from several laboratories suggests that in addition to the activation of positive factors (on- cogenes), the loss of negative regulatory functions (tu- mor suppressor genes) may also be a necessary but
distinct step in neoplastic development (5). This paper
will discuss the detection of activated oncogenes in^ ro-
dent tumors and the implication of oncogenes in^ risk
analysis of carcinogen-induced rodent tumor data.
Detection of Activated Oncogenes
in Tumors
Detection of activated oncogenes in^ neoplasia^ can^ be
achieved by using several different techniques depend-
ing on^ how the^ particular^ oncogene^ might^ be^ activated.
Abnormal expression of oncogenes in tumors due to
amplification of the^ gene may be detected^ by dot^ blot
or Southern blot analysis. Increased expression due to
deregulation of^ the^ gene may be^ detected^ by dot^ blot
as well as Northern blot analysis. Chromosomal trans-
locations may be detected by cytogenetic analysis. Ex-
STOWERS ET AL.
Proto-Oncogene
c-onc
c-myc c-abl
N-myc c-myc Ki-ras
c-Ha-ras c-Ki-ras c-N-ras
Activation
Retrovirus Transduction
Oncogene
v-onc
Chromosomal Ig/myc Translocation bcr/abl
Gene Amplification
Point Mutation
DM/HSR
12th, (^) 13th, 61st Codon Mutation
Examples Acutely Transforming Leukemia/Sarcoma Virus Murine Plasmacytoma Human Burkitt's Lymphoma CML Neuroblastoma Small Cell Lung Carcinoma Adrenocortical Tumor of Mice Colon, Carcinoma Lung Carcinoma AML Chemical Induced Rodent Tumors myc myb erb B mos int- int-
FIGURE 1. Mechanisms of proto-oncogene activation.
amples of^ abnormal^ expression of^ oncogenes detected
in human tumors and tumor cell lines are shown in Table
A number of oncogenes present in human tumors as
well as animal tumors have been detected by the NIH/
3T3 transfection assay. The NIH/3T3 transfection tech-
nique involves the ability of the NIH/3T3 mouse fibro-
blast to accept and express genes from donor tumor
DNA, resulting in the formation of transformed cells.
Only a^ few^ years ago, Shih^ et^ al.^ (6) were^ the first^ to
show that DNA from carcinogen-transformed cell lines
could cause transformation of NIH/3T3 cells after trans-
fection. This transformation was characterized by a
change in the morphology of the NIH/3T3 cells and by
anchorage-independent growth. Other investigators us-
ing this technique were then able to show that dominant
transforning genes or oncogenes were present in hu-
man tumors and in carcinogen-induced animal tumors.
An extension of the NIH/3T3 transfection assay that
affords greater sensitivity is the nude mouse tumori-
genicity assay (7). This involves cotransfection of NIH/
3T3 cells with tumor DNA and a selectable marker
Table 1. Abnormal expression of oncogenes in^ human tumors and tumor cell lines.
Mechanism Tumor type Oncogene Reference Amplification Breast tumor neu^ (24) Amplification Squamous cell^ c-erbB^ (25) carcinoma Amplification Small^ cell^ lung c-myc (26) carcinoma Amplification Small cell^ lung L-myc (27) carcinoma Amplification Neuroblastoma (^) N-myc (28) Amplification Acute (^) myelogenous c-myb (29) leukemia Translocation Chronic myelogenous c-abl (^) (80) leukemia Translocation Burkitt's lymphoma c-myc (31)
gene. The selected cells are then injected SC into the immunocompromised mice. The tumors that develop from the nude mice are then analyzed using the tech- niques described earlier to characterize the activated oncogenes. The majority of activated genes in human tumors de- tected by the NIH/3T3 assay have been members of the ras gene family: the H-ras, the K-ras, and the N-ras. Early studies using the NIH/3T3 assay showed only ras gene activation in a low percentage ofthe human tumors (approximately 10%). Later studies have shown that other oncogenes can also be detected in the human tu- mors by this assay, including the Ica (8), hst (9), and the trk (10) oncogenes. In addition, the percentage of certain tumor types that test positive for activated ras oncogenes are higher than 10%. For example, Verlaan- deVries et al. (11) detected activated ras genes in the 27% of acute myeloid leukemia examined. Ananthas- wamy et al. (12) detected Ha-ras genes in four of six human squamous cell carcinomas examined. The addi- tion of the tumorigenicity parameter to the assay sys- tem appears to improve the efficiency in detection of activated oncogenes in human tumors. A (^) variety of animal tumor model systems have also been examined for activated genes using the NIH/3T assay. These include^ spontaneous tumors in^ rats and mice, tumors^ that^ arise^ after^ single or^ multiple doses of (^) carcinogen, and tumors (^) that arise after long-tern exposure to^ a^ carcinogen. Examples of^ the activated genes in^ the different^ tumor^ model^ systems are^ shown in Table 2. Like the human (^) tumors, the (^) majority of activated (^) oncogenes detected in the animal tumors are members of the ras gene (^) family. Other (^) oncogenes have also been detected in animal tumors using the NIH/3T assay (Table 3). One example is the activated neu on- cogene found in nervous tissue tumors induced in rats by transplacental exposure to N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). The c-raf on-
Promoter/Enhancer P LTR/c-oncLTR/Common Insertion (^) Domain
Chronic, Non- Transforming Leukemia Virus
82
STOWERS ET AL.
Table 3. Non-ras oncogenes detected in human (^) and rodent tumors.
Table 4. Detection of activated K-ras (^) oncogene in tetranitromethane-induced (^) lung tumors in (^) mice and rats.
Tumor Treatment Oncogene (^) Reference Neuroblastomas (R)a ENU neu (38) Schwannomas (R) MNU neu (^) (16) Stomach carcinomas hst (^) (9) (H) Colon carcinomas (H) trk (10) Hepatocellular Untreated raf (23) carcinomas (M) Hepatocellular lca (8) carcinoma (H) Hepatocellular (^) AFB,b (35) carcinomas (R) Hepatocellular Untreated? (23) carcinoma (M) Hepatocellular Furfural? (23) carcinomas (M) Pulmonary Untreated? c adenocarcinoma (M) Nasal (^) squamous MMS (^)? (13) carcinomas (R) Skin carcinoma (M) DMBA? (17) Skin carcinoma (M) DB[c,h]ACR? (17) a (^) Letters in parentheses (^) indicate species in (^) which tumors occur. R, rat; M, mouse; H, human. bAbbreviations: (^) AFBj, aflatoxin B1; (^) MMS, methylmethanesulfo- nate; (^) DB[c,h]ACR, dibenz[c,h]acridine CJU. Candrian and M.W. (^) Anderson, unpublished data.
implies that activation of oncogenes in long-term car-
cinogenic studies in the mouse may or may not be the
result of direct interaction of the chemical with DNA
(U. Candrian, unpublished data; 18). It is possible in
some instances that the chemical did activate the on-
cogene directly and is consistent with the chemical bind-
ing to the DNA. In other instances, the chemical may
have increased the background tumor incidence by a
mechanism such as cytotoxicity or receptor-mediated
promotion. If the pattern of activated oncogenes in the
chemically induced tumors is different from that in the
spontaneously occurring tumors, then the chemical
probably caused the mutations, at least in some of the
tumors. One example in which the chemical's role in
oncogene activation is not known is the activated K-ras
oncogene detected in tetranitromethane (TNM)-induced
rat and mice lung tumors. TNM is a mutagen and an
irritant. However, the interactions between TNM and
DNA are not known. In a recent long-term carcinogen-
esis study conducted by the NTP, chronic exposure to
TNM resulted in a high incidence of primary lung tu-
mors in^ Fischer 344 rats and B6C3F1 mice (19). K-ras
oncogenes with a GGT-)GAT mutation in the 12th codon
were observed in 18 of 19 rat lung tumors and 10 of 10
mouse (^) lung tumors (^) (Table 4). The activation of the K-
ras oncogene in these TNM-induced lung tumors may
be the result of one or more (^) actions of the chemical: a
direct consequence of TNM-induced DNA damage; the
tumors may be spontaneously occurring; enhancement
of spontaneously occurring K-ras by TNM-induced cell
replication; or combination of direct TNM-induced DNA
damage and^ enhancement^ of spontaneous occurrence.
It is a distinct (^) possibility that these (^) activated K-ras
DNA source
Transforming K-ras gene with GGT-*GAT mutation in 12th codon,a number (^) positive/number tested Rat adenocarcinoma 12/12 (^) (100%) Rat squamous cell carcinoma 3/4 (^) (75%) Rat (^) adenosquamous carcinoma 3/3 (100%) Mouse (^) adenocarcinoma 8/8 (100%) Mouse adenoma 2/2 (100%) aThe presence of transforming genes was detected by NIH/3T transfection and/or (^) oligonucleotide hybridization, and the frequency of (^) transforming genes is (^) represented by the numbers in (^) parentheses.
oncogenes with^ GC--AT transitions in the second base of the 12th codon (^) are spontaneous, since an activated K-ras with the same mutation was observed in a spon-
taneously occurring pulmonary adenocarcinoma in the
B6C3F1 mouse (U.Candrian, unpublished (^) data). Even
though spontaneous lung tumors in the Fischer 344 rat
were not observed in this study, it (^) is still possible that the irritant property of TNM could have promoted the
cells, which activated the K-ras or enhanced the spon-
taneously occurring K-ras. The reproducible (^) detection of the K-ras in lung tumors of mice and (^) rats suggests that TNM could have directly induced the mutation. (^) In
support of this conclusion, mutagenicity studies have
shown that TNM causes mutant bacterial strains to (^) rev- ert to the wild type by the same GC-*AT transition.
Studies on the possible interactions of TNM with DNA
are required to precisely determine the origin of the
activated K-ras oncogenes in these TNM-induced lung
tumors.
Although gene amplification and chromosomal trans-
location have been observed in several types of human
tumors, these activating mechanisms have not been ex-
tensively observed or studied in spontaneous or chem- ically induced rodent tumors. Sawey et al. (20) did ob-
serve c-myc gene amplification and restriction poly-
morphisms in addition to activated K-ras genes in rat
skin tumors induced by ionizing radiation. Quintanilla
et al. (21) suggested that amplification of the mutated H-ras gene (^) may be (^) involved in the progression of mouse skin (^) papillomas to (^) carcinomas. Further studies are re- quired to determine (^) the possible role of chemicals and
radiation in the activation of proto-oncogenes by gene
amplification, chromosomal translocation, and other
mechanisms that can alter (^) gene expression.
Carcinogen-induced rodent tumor models may be use-
ful in determining the temporal activation of oncogenes
in tumor development. Evidence in several animal stud-
ies suggests that activation of the ras proto-oncogene
is an early event. The activated ras gene has been de-
tected in many benign tumors, including mouse skin
papillomas, mouse lung and liver adenomas, and basal
cell and clitoral gland tumors of the rat. This implies
that the activated ras was present in the cell that clon-
ally expanded to these benign tumors. In addition, it
was recently shown that mouse epidermal cells injected
84
ROLE OF ONCOGENES IN CHEMICAL (^) CARCINOGENESIS 85
in vivo with the viral Ha-ras gene can be promoted with
12-0-tetradecanoyl-phorbol-13-acetate (TPA) to papil-
lomas (22). Thus, activation of the ras proto-oncogene
may be the initiation event in some model systems. Moreover, dormant initiated cells with the activated ras gene can survive surrounded by normal cells until stim- ulated to proliferate by some endogenous or exogenous agent.
Implications for Risk Analysis
The number of proto-oncogenes that must be acti-
vated in order to convert a normal (^) cell into one that is tumorigenic is unknown at (^) present. However, there is increasing evidence that the transformation of a (^) normal cell into a tumorigenic cell involves the activation and concerted expression of several (^) proto-oncogenes as well as, perhaps, the inactivation of suppressor genes. (^) Con- tinued research on mechanisms of (^) oncogene activation in animal and in vitro models may provide new insights into several long-standing problems in chemical carcin-
ogenesis and risk analysis of carcinogenesis data.
Oncogene analysis on tumors from long-term carcin-
ogenesis studies that are employed to help identify po-
tential human carcinogens can be useful in several ways. The analysis can help identify chemicals that can acti-
vate proto-oncogenes in vivo to cancer-causing genes.
Model systems very susceptible to chemically induced
tumors, such as the B6C3F1 mouse liver tumors, appear to be suited for this purpose (23). The classification of
chemicals as initiators, promoters, complete carcino-
gens, etc., may become clearer as we better understand the sequential requirements for activation of oncogenes in the various animal model and cell culture systems. In (^) particular, comparison of patterns of oncogene ac-
tivation in^ spontaneously occurring and chemical-in-
duced tumors should assist in^ determining mode(s) of
action of a carcinogen. Low-dose and species-to-species
extrapolation ofrisk from carcinogenic data may become
more reliable from examination of (^) oncogene activation and expression in animal model (^) systems for carcino-
genesis. These and similar approaches to explore the
mechanisms by which chemicals induce tumors in (^) animal
model systems may remove some of the uncertainty in
risk analysis of rodent carcinogenic data.
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